Methods for expressing nucleic acid sequences using nucleic acid constructs comprising hypoxia response elements

ABSTRACT

Nucleic acid constructs comprising hypoxia response elements in operable linkage with a coding sequence of a gene of interest, and methods for expressing a nucleic acid sequence using the constructs, are disclosed. In particular, such nucleic acid constructs comprise genes encoding prodrug activation systems or cytokines.

This application is a divisional of U.S. Ser. No. 08/693,174, filed Dec.12, 1996, now U.S. Pat. No. 5,942,434, which is a 371 applicationPCT/GB95/00322, filed Feb. 15, 1995.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention is concerned with hypoxically-inducible expressioncontrol sequences, nucleic acid constructs comprising such sequences,and their use for selective targeting of anti-cancer therapy and otherkinds of therapy where target cells are affected by hypoxia.

2. Detailed Description of the Related Art

Vile and Hart (1993) describe a method in which certain gene promoters,which are preferentially active in melanocytic cells, were used todirect gene expression of a reporter gene specifically to melanoma cellsin vitro, and in vivo in mice. Constructs consisting of the promotersand the beta-galactosidase gene were directly injected into mice and thereporter gene was expressed in melanoma cells and in some normalmelanocytes but not in surrounding normal tissue. However,tissue-specific promoters will necessarily be limited in the tumoursthat they can target and will also be liable to target normal cells ofthe tissue concerned (as was noted in Vile and Hart above).

Cancers tend to outgrow the blood supply and often have areas of hypoxiaand necrosis which distinguish them from normal tissue. This featurealso makes tumours resistant to radiation due to low oxygen levels andx-ray treatment becomes less effective. Certain genes such as the genefor erythropoietin, are known to be regulated by hypoxia. Erythropoietinis a hormone which regulates erythropoiesis and hence blood oxygencontent. Cis-activating DNA sequences that function as tissue-specifichypoxia-inducible enhancers of human erythropoietin expression have beenidentified (Semenza et al, 1991). A DNA enhancer sequence located 3′ tothe mouse erythropoietin gene has been shown to confer oxygen-regulatedexpression on a variety of heterologous promoters (Pugh et al, 1991). Ithas further been demonstrated that the oxygen-sensing system whichcontrols erythropoietin expression is widespread in mammalian cells(Maxwell et al, 1993).

A second example of a hypoxia-associated regulator is a regulator whichlies 5′ to the mouse phosphoglycerate kinase gene promoter. The sequenceof the regulator has been published (Mcburney et al, 1991) but itshypoxia inducible properties have not previously been considered ordefined in the literature. It has now been recognised by the inventorsthat the native sequence of the regulator has hypoxically-induciblefeatures. The nucleotides responsible have been defined and theinventors have shown that repeating the sequence leads to increasedinduction of the gene whose expression is controlled. Further, theinventors have shown that using the interleukin-2 gene undertissue-specific promoters is an effective strategy for specifictargeting of tumours.

There are anti-cancer drugs that become activated under hypoxia (Workmanand Stratford, 1993), but the use of a drug activation system where theenzyme activating the drugs is greatly increased under hypoxia willprovide a far superior therapeutic effect.

SUMMARY OF THE INVENTION

The invention provides a nucleic acid construct comprising at least onegene encoding a species having activity against disease, operativelylinked to a hypoxically inducible expression control sequence.

When the construct is present in a suitable host cell, expression of thegene will thus be regulated according to the level of oxygenation.Preferably the expression control sequence is a promoter or enhancer. Ina host cell under hypoxic conditions, expression of the gene will beinitiated or upregulated, while under conditions of normoxia (normaloxygen level) the gene will be expressed at a lower level or notexpressed at all. The expression level may vary according to the degreeof hypoxia. Thus, a gene product which has therapeutic activity can betargetted to cells affected by disease, eg. tumour cells.

The species encoded by the gene in the construct according to theinvention may be for example a cytokine, such as interleukin-2 (IL-2)which is known to be active in the immune response against tumours.Genes encoding other molecules which have an anti-tumour effect may alsobe used.

In a preferred embodiment of the construct according to the invention,the species encoded by the gene is a pro-drug activation system, forexample the thymidine phosphorylase enzyme, which converts a relativelyinactive drug into a much more potent one. Transfection of the thymidinephosphorylase gene into human breast cancer cells has been shown togreatly increase the sensitivity of the cancer cells to 5-deoxy-5FU (seeExample 8). The thymidine phosphorylase gene has not previously beenreported as an agent for gene therapy. Another pro-drug activationsystem which can be used is cytosine deaminase, which activates thepro-drug 5-fluorocytosine (5-FC) to form the antitumour agent5-fluorouracil (5-FU). A further example of a pro-drug activation systemfor use in the invention is cytochrome p450 to activate the drug SR4233(Walton et al, 1992).

The construct according to the invention may contain more than one geneand more than one type of gene. Additional genes may encode furtherspecies having activity against disease, or they may have gene productswith other activities.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D. FIG. 1A shows the structure of the test plasmids, where Erepresents Epo enhancer sequence, SV40P represents the SV40 earlypromoter and GH represents the body of the growth hormone gene. FIGS.1B-D shows the independent experiments measuring hypoxic inducibleactivity of the constructs comprising the Epo enhancer sequence.

FIGS. 2A-2B. FIG. 2A shows the partial nucleotide sequence of the 253 bpEcoR1-Spe1 fragment containing the enhancers (EMBL accession No. M18735,nucleotides 417 to 676). The position of the deletions (D) from the 5′end of the sequence are marked by arrows. FIG. 2B shows the ratio ofhypoxic to normoxic expression conferred on the reporter by EcoR1-Spe1fragment and deletion thereof Values represent means of three separatetransfections, with bars showing SEM.

FIG. 3 shows a survival curve for the parent cell line MCF-7 in breastcancer (WT) and a derivative from it that has been transfected with agene for thymidine phosphorylase, MCF-7(−4).

FIG. 4 shows the drug sensitivity results of transfectant (−4) to5-deoxy 5FUdR as compared to the wild-type.

FIG. 5 shows the drug sensitivity results of clones −4, −7, −12, −16,and +4 to 5-deoxy 5FUdR compared to the wild-type.

FIG. 6 shows the drug sensitivity results of clones −4, −7, −12, −16,and +4 to 5FU.

FIG. 7 shows the drug sensitivity results of the mixed cell population(−4:wt) to 5-deoxy 5FUdR.

FIGS. 8A-8C. FIG. 8A shows CD2 induction data for M3. FIG. 8B shows CD2induction data for sTK5. FIG. 8C shows CD2 induction data for 9-3C. InFIGS. 8A-8C, the CD2 induction data was measured with oxygen at 5%, 2%,1%, 0.001% (N₂) almost no oxygen), 0% O₂ (AnO₂). n represents the numberof experiments performed. CD2 expression was measured immediatelyfollowing treatment (Oh) and five hours post-treatment (5h).

FIGS. 9A-9B. FIG. 9A shows drug sensitivity results of transfected cells9-3C and M3 to 5-FU post-hypoxia. FIG. 9B shows drug sensitivity resultsof transfected cells 9-3C and M3 to 5-FC post-hypoxia.

Hypoxically-inducible promoters or enhancers may be chosen from thosereferred to herein, or they may be other hypoxically-inducible promotersor enhancers. It is anticipated that other hypoxically-induciblepromoters or enhancers will be discovered; oxygen-sensing systems arewidespread in mammalian cells and many genes are likely to be underhypoxic control.

Preferably, the nucleic acid construct according to the inventioncomprises at least one hypoxia response element which confers hypoxiainducibility on the expression control sequence. There may be forexample two or more hypoxia response elements linked so as to increasehypoxia inducibility and thus to increase the induction of the gene orgenes under hypoxia. Hypoxia response elements may be chosen from amongthose referred to herein, or they may be other hypoxia responseelements. As noted above, oxygen-sensing systems are widespread inmammalian cells, and it is expected that other hypoxia response elementswill be found.

The following hypoxia response elements may be used in the constructaccording to the invention:

a). The following portion of the transcriptional enhancer lying 3′ tothe mouse erythropoietin (Epo) gene:

GGG CCC TAC GTG CTG CCT CGC ATG G (25) [SEQ ID NO: 1]′

b) One of the following portions of the 5′ flanking sequence of themouse phosphoglycerate kinase (PGK) gene:

CGC GTC GTG CAG GAC GTG ACA AAT (P24) [SEQ ID NO: 2] or GTC GTG CAG GACGTG ACA (P18) [SEQ ID NO: 3]

These PGK sequences have not been previously recognised as havinghypoxically-inducible properties.

All of these sequences have counterparts in human genes and are highlyconserved between species. They are also well characterised.

The invention therefore also provides a hypoxically inducible expressioncontrol sequence which comprises the nucleic acid sequence:

CGC GTC GTG CAG GAC GTG ACA AAT (P24) [SEQ ID NO: 2] or GTC GTG CAG GACGTG ACA (P18) [SEQ ID NO: 3]

or a nucleic acid sequence with substantial homology thereto. Thesesequences can be found in EMBL database, accession no. M18735, atnucleotides 631 to 654 and 634 to 651.

The construct according to the invention may comprise more than one eg.three or more copies of one of the Epo or PGK sequences given above.Additionally or alternatively, a longer portion of the Epo or PGK-1enhancer or flanking sequence may be used in the construct, which longerportion comprises the hypoxia response element and part of thesurrounding sequence.

Hypoxically-inducible expression control sequences and hypoxia responseelements may be chosen so as to be operative in particular tissues orcell types to be targetted therapeutically, or they may be chosen towork in a wide range of tissues or cell types.

The invention further provides a nucleic acid construct as describedherein for use in the treatment of a patient suffering from a disease inwhich hypoxia is a cause or a symptom or is otherwise present.Alternatively, the invention provides a method of treatment of a patientsuffering from a disease in which hypoxia is a cause or a symptom or isotherwise present, which method comprises administering to the patient anucleic acid construct as described herein.

The nucleic acid constructs will be useful for treating cancer patients.In hypoxic tumour cells, the physiological stimuli will be such that thegene which has activity against the disease is expressed. Although itmay be very difficult to get all cells to take up the gene to switch iton, experiments show that even with 10% of cells expressing thethymidine phosphorylase gene there is an effect on other cells whichresults in a 10-fold increase in sensitivity of the whole population.This is known as the bystander effect and is probably due to activemetabolites of the anticancer drug passing from one cell to another.Since this drug kills proliferating cells, it should still have muchless toxicity on normal tissue than on cancer cells. Sometimes, nearbynormal cells will also express the gene and the active drug or cytokineor other species encoded by the gene will be able to diffuse from thenormal cells into the tumour.

The inventors have demonstrated both stable and transient transfectionof cells with genes under the control of hypoxically inducibleenhancers. Transient transfection only lasts for a few days, whereasstably transfected genes insert into the cell genome and can persistindefinitely. Stable transfection may prove to be necessary fortherapeutic applications of the invention but it is possible thattransient transfection will be sufficient.

Administration of constructs according to the invention for therapeuticpurposes can be by injection of DNA directly into the solid tumour.Certain types of cell including tumour cells, skin cells and musclecells take up naked DNA, and tumour cells do so particularly well. Theconstructs may thus be administered in the form of naked DNA plasmids.Alternatively other vectors such as retroviruses may be used.

A suitable therapeutic regime will be direct injection of DNA into theaffected site, and administration of pro-drug in the case of a constructencoding a pro-drug activation system, optionally combined withradiotherapy.

Although the invention is described above in relation to targeting oftumour cells, it will also be useful in other types of disease wherehypoxia occurs, including for example coronary artery disease andstrokes. The nucleic acid construct may comprise a gene encoding apro-drug activation system for a suitable drug or the gene may encode acytokine or a growth factor. A vascular growth factor can be used tostimulate new blood vessel formation in hypoxic areas and expression ofthe growth factor by the construct will then be automatically switchedoff when the area becomes revascularised.

The inventors have carried out deletional analysis and mutationalanalysis of the mouse Epo enhancer sequence, in the cell lines HepG2 anda23 (a23 are non-Epo producing cells). Transient tranfection experimentswere performed using plasmids containing full or partial enhancersequences lying 1.4 kb 5′ to the α₁ globin reporter gene.

Three critical sites for enhancement were defined, corresponding to thenucleotides 5-12, 21-24 and 33-34 of the 96 nucleotide enhancer sequence(mouse Epo enhancer: EMBL accession no. X73471). All three regions wereabsolutely necessary for enhancer function in the above experiments. Butoverall, studies indicated that the 1-25 or 1-26 nucleotide sequence hashypoxically inducible operation (EMBL, X73471, nucleotides 407 to 431 or432. Inducible operation via the 1-26 nucleotide sequence was alsodemonstrated for MEL and HeLa cells, which have previously been foundunable to support oxygen-regulated reporter gene expression usingplasmids containing the 1-96 nucleotide enhancer sequence (Maxwell et al1993). A total of 19 cell lines (some unreported) have now been testedby the inventors and none has been found to lack the capacity tomodulate oxygen dependent changes in reporter gene expression by thesequence 1-26. Thus, whereas it was previously suggested that thehypoxically inducible response was not universal in mammalian cells, itnow seems more likely that it is.

Recent studies of the human Epo enhancer also define at least threecritical regions of the sequence (Semenza et al 1992 and Blanchard et al1992).

The examples which follow contain experimental demonstrations by theinventors which indicate the manner in which different aspects of theinvention work.

EXAMPLES Example 1 Action of Subsequences from the Mouse Epo Enhancer onan Adjacent SV40 Promoter

FIG. 1 (A) shows the structure of the test plasmids, where E representsEpo enhancer sequence, SV40P represents the SV40 early promoter and GHrepresents the body of the growth hormone gene. The Epo enhancersequence (E) used in each test plasmid is indicated beneath thecorresponding bars of the histogram. A co-transfected plasmid containingthe α₁ globin gene alone (pBSα⁻) was used to correct for transfectionefficiency. Expression is normalised to that of the enhancerless pSVGHin normoxic cells and represents the mean± S.D. of three independentexperiments (B). Inducible activity is conveyed by sequence 1-25 in allcell types. Inducible activity was much increased by concatamerizationof this sequence which also conveyed some constitutive activity innormoxic cells (1-25)₂ and (1-25)₅. In contrast, neither constitutivenor inducible activity was conveyed by sequence 25-60 in any cell type.Concatamers of this sequence were tested in either orientation (25-60)₃and (60-25)₄ but none had any action. The monomeric sequence 1-60 wasmore active than the monomeric sequence 1-25 in HepG2 cells and a23 butnot MEL cells.

Example 2 Oxygen-Regulated Control Elements in the PGK-1 and LDH-A Canes

The materials and methods below are employed in Examples 3-6.

Cell Lines and Culture Conditions

The cell lines used were HepG2 (human hepatoma), HeLa (human cervicalcarcinoma) and L cells (mouse fibroblast) grown in minimal essentialmedium, with Earle's salts, supplemented with fetal bovine serum (10%),glutamine (2 mM), penicillin (50 units/ml) and streptomycin sulfate (50μg/ml). For assay of endogenous gene expression, and for preparation ofnuclear extracts, cells were grown to approximately 70% confluence. Themedium was then replaced, and cells subjected to the followingconditions for 14-16 hours: (1) normoxia (20% oxygen, with 5% CO₂ and75%N₂); (2) hypoxia (1% oxygen, with 5% CO₂ and 94% N₂ in Napco 7100incubator); (3) hypoxia with cycloheximide (100 μM); (4) normoxia withcobaltous chloride (50 μM); (5) normoxia with cyanide (100 μM) ; (6)hypoxia with cyanide (100 μM).

Transient Transfection

In all experiments the test plasmid (10-100 μg), containing either humanα₁ globin (α) or human growth hormone (GH) as a reporter, wascotransfected with a control plasmid (10-50 μg) using electroporation aspreviously described (Pugh et al. 1991). The control plasmid, whoseexpression was not altered by hypoxia, was either the human α₁ globingene, when the test plasmid contained GH; or FGH, a fusion geneconsisting of the mouse ferritin promoter (290 bp) linked to the humangrowth hormone gene, when the test plasmid contained the α globin gene.

Details of test plasmid design are as follows. The mouse PGK-1 5′flanking sequence used in the basic construct (pPGKGH) was a 502 bpfragment of the PGK-1 sequence in pDEneo, extending from the EcoR1 siteat −523 bp (1=translational initiation) to the Taql site at −21 bp. Thenucleotide sequence of the mouse PGK-1 gene is shown in SEQ ID No. 4,which is the same sequence as found in EMBL database, accession no.M18735 mentioned above. The translation start of the sequence isnucleotide 946. Accordingly, −523 bp to −21 bp correspond to nucleotide423 to nucleotide 925 of SEQ ID No. 4. The mouse LDH-A gene sequence inpLDHGH was generated by PCR amplification from mouse genomic DNA of a233 bp fragment (−186 to +47 from transcriptional start site) usingoligonucleotides derived from published sequence. The nucleotidesequence of the mouse LDH-A gene is shown in SEQ ID No. 5. Thetranscription start of the sequence is nucleotide 1118. Accordingly,−186 bp to +47 bp correspond to nucleotide 932 to nucleotide 1164 of SEQID No. 5. Plasmid DNA for transfection was purified on a caesiumgradient; nucleotide sequence of the crucial elements of all plasmidswas confirmed by direct sequencing.

After electroporation transfected cells were spilt equally and incubatedin parallel for 14-16 hours in 8 ml of culture medium in 100 mm Petridishes under normoxic or hypoxic conditions, or exposed to chemicalagents, as described above.

RNA Analysis

RNA was extracted, analysed by RNase protection, and quantitated aspreviously described (Pugh et al 1991). For assay of transientlytransfected material 3-10 μg of RNA was subjected to doublehybridisation with probes which protected 120 bp of GH mRNA and either132 bp (α132) or 97 bp (α97) of α globin mRNA, depending on whether thetest or control plasmid contained α globin.

For assay of endogenous PGK-1 gene expression 50 μg of RNA extractedfrom HepG2 cells was hybridised with a riboprobe which consisted of 121bp from the 5′ end of the exon 3 of the human PGK-1 gene, together with68 bp of adjacent intron (obtained by PCR of genomic DNA and cloning bystandard methods). For assay of endogenous LDH-A gene expression 25 μgof RNA obtained from L cells was hybridised with a riboprobe whichconsisted of 47 bp from the 5′ end of exon 1 of the mouse LDH-A gene,together with adjacent 5′ flanking sequence (obtained by PCR of genomicDNA and cloning by standard methods). In these experiments a smallquantity (0.5 μg) of RNA extracted from K562 cells (a human Epo cellline which expresses a globin mRNA abundantly) was added to each samplebefore hybridisation, and the samples were probed concurrently with theα97 probe in addition to the PGK-1 or LDH-A probe. This provided a meansof determining that sample processing and gel loading was comparablebetween specimens.

Nuclear Extract

Cells were cooled rapidly, harvested into ice cold phosphate bufferedsaline, and pelleted at 2000 g. A modification of Dignam's protocol wasused to prepare nuclear extract. Briefly, cells were swollen for 10minutes in Buffer A (10 mM Tris-HCl pH 7.4, 10 mM KCl, 1.5 mm MgCl₂)lysed using a Dounce homogenizer, and extracted for 30 minutes in BufferC (20 EM Tris-HCl, pH 7.4, 420 mM KCl, 1.5 mM MgCl₂, 20% glycerol).Nuclear debris was pelleted at 10000 g and the supernatant dialysed for2 hours against Buffer D (20 mM Tris-HCl pH 7.4, 100 mM KCl, 0.2 mMEDTA, 20% glycerol). The entire procedure was performed at 4° C. usingprecooled buffers. In addition buffers A and C containedphenylmethysulfonyl fluoride, 0.5 mM; aprotinin, 1 μg/ml; leupeptin, 1μg/ml; pepstatin, 1 μg/ml; sodium orthovanadate, 1 mM; benzamidine, 0.5mM; levamisole, 2 mM; β glycerophosphate, 10 mM; DTT, 0.5 mM. Sodiumorthovanadate and DTT were also added to buffer D. Following dialysis,aliquots of nuclear extract were frozen in a dry ice/ethanol bath andstored at −70° C.

Electrophoretic Mobility Shift Assays

Oligonucleotides used as probes or competitors were purified bypolyacrylamide gel electrophoresis. Labelling was performed with{gamma-³²P}ATP (300 Ci/mmol) using T₄ polynucleotide kinase. Labelledoligonucleotides were annealed with 4× molar excess of the complementarystrand. Unlabelled oligonucleotides were annealed in molar equivalentquantities.

The binding reactions were performed in a 20 μl volume containing 50 mMKCl, 1 mM MgCl₂, 0.5 mM EDTA, 5 mM DTT, 5% glycerol, and 0.15-0.30 μg ofsonicated polydldc. Nuclear extract (5 μg unless otherwise stated) wasincubated with this mixture for 5 minutes at room temperature, beforeprobe (approximately 0.5 ng)and specific competitors were added.Incubation was continued for a further 10 minutes. Reactions wereelectrophoresed (12.5 v/cm) at 4° C. using 5% polyacrylamide in 0.3×TBE(pH 7.3 at 4° C.).

Example 3

RNA Analysis of PGK-1 and LDH-A Gene Expression

Expression of the endogenous human PGK-1 gene in HepG2 cells and of theendogenous mouse LDH-A gene in L cells was assayed by RNA analysis.Cells were exposed for 16 hours to normoxia (20% O₂) hypoxia (1% O₂) andcycloheximide 100 μM, normoxia and cobaltous chloride 50 μM, normoxiaand cyanide 100 μM, hypoxia and cyanide 100 μM. 50 mg RNA was used foreach hybridisation reaction. For each gene, both hypoxia and exposure tocobalt led to a 2-3 fold induction of gene expression. The proteinsysthesis inhibitor cycloheximide abrogated the response to hypoxia.Cyanide did not affect the hypoxic response, neither inducing expressionin normoxia, nor preventing induction by hypoxia.

Example 4 Location of Sequences Responsible for Hypoxia InducibleExpression of the Endogenous PGK-1 and LDH-A Genes

Portions of the 5′ flanking sequence of PGK-1 and LDH-A genes werelinked to a growth hormone receptor gene. Expression in transfectedHepG2 cells of fusion genes containing either a 502 bp fragment from thePGK-1 enhancer/promoter region (pPGKGH) or a 233 bp sequence from theLDH-A promoter (pLDHGH), was measured after exposure to conditions ofhypoxia, normoxia etc. as used in Example 3. In each case hypoxicallyinducible expression was conveyed by the sequences, a somewhat greaterlevel of inducibility being observed than for the respective endogenousgene. The inducible operation of these sequences contrasted with theSV40 virus promoter, the α globin promoter, the ferritin promoter andthe herpes simplex thymidine kinase promoter, none of which had thisproperty. Responses were not specific for HepG2 cells and similarinducible responses were obtained in transfected HeLa cells.

In this example, 5 μg of RNA was used for each hybridisation reaction,and the α132 and GH probes were used to detect expression of thetransfection control and test plasmids respectively.

Example 5 Deletional Analysis of the PGK-1 Enhancer Region

FIG. 2 shows (A) partial nucleotide sequence of the 253 bp EcoR1-Spe1fragment containing the enhancers (EMBL accession no. M18735,nucleotides 417 to 676). The positions of deletions (D) from the 5′ endof the sequence are marked by arrows. FIG. 2 (B) shows the ratio ofhypoxic to normoxic expression conferred on the reporter by EcoR1-Spe1fragment and deletion thereof. Values represent means of three separatetransfections, with bars showing SEM.

A series of nested deletions was carried out as indicated in FIG. 2. Thegreatest reduction in the amplitude of hypoxic induction occurred withdeletion of 20 bp between D9 and D11. The functional importance of thisregion was confirmed by the finding that a pGKGH fusion gene from which18 bp between the positions of D9 and D11 had been removed no longerdemonstrated hypoxic inducibility.

Example 6 Functional Analysis of the Oxygen Regulated Element of the PGKEnhancer

For analysis of the functional characteristics of this isolated sequence(the 18 bp sequence described in Example 5), oligonucleotides werecloned into a site 10 bp 5′ to the TATA box of the herpes simplexthymidine kinase promoter in a thymidine kinase—growth hormone fusiongene. The 18 bp element (P18) whose deletion abrogated hypoxic inductionof the PGKGH construct, was capable of conferring responsiveness tohypoxia when placed in either orientation. Concatamers operated morepowerfully than monomers. Extension of P18 to a 24 bp element (P24) didnot lead to increased activity.

Example 7 Stable Transfection of Cells with the PGK-1 Hypoxic Promoter

HeLa cells were transfected by electroporation with supercoiled plasmidDNA. Stably transfected cells were selected by growth in G418 medium.Two plasmids were transfected. The first contained a hypoxic promotercoupled to the gene for the cell surface marker CD2 and the secondcontained the same hypoxic promoter coupled to the gene for the enzymecytosine deaminase as well as a constitutively acting promoter (SV40early region promoter) coupled to a neomycin resistance region. Thehypoxic promoter used in this transfection experiment consisted of a 456bp Sph-1/Taq-1 fragment from the mouse PGK-1 5′ flanking sequence intowhich 3 copies of the active sequence P24 had been inserted at the Spe-1site.

This system was designed in order to enable us to create signal pathwaymutant cells. Essentially the CD2 and cytosine deaminase genes are beingused as selectable markers and the neomycin resistance gene provides ameans of retaining the transfected plasmid and therefore selecting cellswhich have lost the response rather than the transfected genes. It isclear that the principle of selectable markers can be used to killunwanted cells, according to the invention.

After transfection cells were grown for two weeks and pools were subjectto a 16 hour incubation at either 21% oxygen (normoxia) or 1% oxygen(hypoxia). Marker gene expression was quantified by RNA analysis.Approximately 10 fold increased expression was observed at 1% oxygenversus 21%. Thus, the selectable marker genes were under the control ofthe hypoxically inducible promoter.

Example 8 Transfection of Human Breast Cancer Cell Line with ThymidinePhosphorylase Gene

Human breast cancer cells of the cell line MCF-7 were transfected with agene for thymidine phosphorylase. Thymidine phosphorylase can convert arelatively inactive drug into a much more potent product. The gene issuitable for linking up with hypoxically-inducible promoters/enhancers.

As a general principle, when genes are transfected into cancer cellsthey will be expressed at different levels and so it is important tostudy several different clones of the cells to see the variability.

Results of drug sensitivity experiments are shown in FIGS. 3-7. −7, −4,−12, +4 and +16 are all derivatives of MCF-7 which have been transfectedwith the thymidine phosphorylase gene.

FIG. 3 shows a survival curve for the parent cell line MCF-7 in breastcancer (WT) and a derivative from it that has been transfected with agene for thymidine phosphorylase. There is relatively little differencein their sensitivity to the drug 5FU. However, the transfectant (−4) isnearly 200 fold more sensitive to 5-deoxy 5FUdR (FIG. 4).

FIG. 5 shows clones −12 and −4 are particularly sensitive to the drug5-deoxy 5FUdR compared to the wild-type. Two other transfectants −7 and+4 and −16 are a little bit more sensitive than the wild-type. Note thatthere is a log scale and therefore the IC50 (the concentration of drugrequired to inhibit growth by 50%) is more than 100 times lower for thetransfectants −12 and −4. Therefore, the expression of one gene at ahigher level can make these cells 100 times more sensitive tochemotherapy. Clearly there is variation depending on the level ofexpression and therefore we want to be able to switch on as high aspossible. The pro-drug 5-deoxy 5FUdR is broken down to 5FU and acomparison of the sensitivity of the cells to 5FU in FIG. 6 reveals thatthere is really very little difference. This means that it is theactivation step that is important rather than downstream events.

Finally, when a drug has been activated by the cancer cells it could betransferred to other cells which would normally be resistant but theactive form of the drug can overcome this. To demonstrate, the sensitivecells (−4 cells) and the wild-type cells were mixed together, and it canbe seen from FIG. 7 that the IC50 drops from 11.6 μM to 1.5 μM i.e. onelog 10-fold increase in sensitivity with only 20% of the cellsexpressing the drug activation gene. Thus, in gene therapy it will notbe necessary to get all of the cells to express the gene, only perhaps20%.

This demonstrates that the target gene thymidine phosphorylase is usefulas a mechanism of activating an anticancer drug and that overexpressingit in even a proportion of cells can confer drug sensitivity, not onlythese cells but adjacent cells.

Example 9 Stable Transfection of Cells with PGK-1 Hypoxically InducibleElements and Different Promoters

The PGK-1 hypoxia response element was evaluated within the context ofthe PGK-1 gene promoter (M3), the 9-27 gene promoter (9-3C) and thethymidine kinase gene promoter (sTK5).

The constructs were similar to those described in Example 7. Thepromoters and PGK-1 hypoxia response elements were placed upstream ofgenes encoding the endothelial CD2 surface protein and the negativeselectable marker cytosine deaminase. The 9-27 and thymidine kinasepromoters were each used with 3 copies of the P18 PGK-1 element and thePGK-1 gene promoter was used with 3 copies of the P24 PGK-1 element.

The stable transfectants were tested for their response to severehypoxia (0.001% and 0% O₂) and different levels of oxygen (0.1, 1, 2, 5and 20% O₂). Cell-surface expression of transfected CD2 was analysedusing fluorescence-labelled anti-CD2 antibodies. Labelled cells wereassayed for CD2 expression on a fluorescence activated cell sorter.

The increase in CD2 production depended on the length and severity ofhypoxia. Following severe hypoxia, CD2 expression in the transfectedlines only increased following subsequent aeration (maximal at 5 hourspost hypoxia), whereas, following less severe hypoxia conditions, CD2expression peaked immediately after hypoxia.

1% O₂ induced CD2 expression, but not to the same degree as severehypoxia (<5 ppm). 5% and 20% O₂ did not result in any CD2 induction.

CD2 expression persisted for 24 hours following reoxygenation.

FIGS. 8a, b and c show CD2 induction data for M3, sTK5 and 9-3Crespectively, with oxygen at 5%, 2%, 1%, 0.001% (N₂, almost no oxygen),0% O₂ (AnO₂). n represents the number of experiments performed. CD2expression was measured immediately following treatment (Oh) and fivehours post-treatment (5h).

Example 10 Hypoxia-Induced Pro-Drug Sensitisation

The bacterial enzyme cytosine deaminase catalyses the conversion ofcytosine to uracil, and can therefore change the pro-drug5-fluorocytosine (5-FC) to the antitumour agent 5-fluorouracil (5-FU).

Transfected cells (9-3C and M3) as described in Example 9 were testedfor sensitivity to 5-FC. Cells were subjected to hypoxic exposure (16hours), followed by exposure to 5-FC or 5-FU (24 hours), followed by a96 hour growth delay.

Results are shown in FIGS. 9a and b (Wt represents non-transfectedHT1080 cells). M3 and 9-3C cell lines were significantly more sensitiveto 5-FC post-hypoxia (FIG. 9a). There was no significant difference in5-FU sensitivity in control (Wt) or transfectant cells following hypoxicor normoxic treatment (FIG. 9b).

Example 11 In vivo Experiment in Mice

Tumour cells (HT1080 cells) were transfected with a DNA constructcontaining the CD2 gene linked to a promoter containing the PGK-lhypoxia response element (as described in Example 9). About a milliontransfected cells were implanted under the skin of a mouse. The tumourwas later excised and histologically sectioned. Staining with anti-CD2antibodies indicated hypoxically-induced expression of the marker gene.

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5 1 25 DNA Murinae gen. sp. 1 gggccctacg tgctgcctcg catgg 25 2 24 DNAMurinae gen. sp. 2 cgcgtcgtgc aggacgtgac aaat 24 3 18 DNA Murinae gen.sp. 3 gtcgtgcagg acgtgaca 18 4 1110 DNA Murinae gen. sp. 4 ggatccacggcctctggccg cattttacca ctgagctaca ctcccaaagc agtcgaaatc 60 acagtggcccaggattgaaa tgatcactta gatgctttgc agtcttgata agacactaaa 120 tctttgtctatcagttactt catctttaat aacagaacgt acttaggaat tttatgagca 180 ttgttagttagcatgacaca tgctatatgt attcgtcatt atgaataatg taaccacagc 240 aattacattgtactttttat tataaaaggg gggaggggaa ggccctggtc cttttttaac 300 ttctgagaggtttcgattac taagtaagac cttatgtaga cttccatttg ggagctgaga 360 aagcagaggattccaaaagg ggatgacatt tgcaaaggtc tagaaaaggc gcctgggaat 420 tctaccgggtaggggaggcg cttttcccaa ggcagtctgg agcatgcgct ttagcagccc 480 cgctggcacttggcgctaca caagtggcct ctggcctcgc acacattcca catccaccgg 540 taggcgccaaccggctccgt tctttggtgg ccccttcgcg ccaccttcta ctcctcccct 600 agtcaggaagttcccccccg ccccgcagct cgcgtcgtgc aggacgtgac aaatggaagt 660 agcacgtctcactagtctcg tgcagatgga cagcaccgct gagcaatgga agcgggtagg 720 cctttggggcagcggccaat agcagctttg ctccttcgct ttctgggctc agaggctggg 780 aaggggtgggtccgggggcg ggctcagggg cgggctcagg ggcggggcgg gcgcccgaag 840 gtcctccggaggcccggcat tctcgcacgc ttcaaaagcg cacgtctgcc gcgctgttct 900 cctcttcctcatctccgggc ctttcgacct cacggtgttg ccaaaatgtc gctttccaac 960 aagctgactttggacaagct ggacgtgaag gggaagcggg tcgtgatgag gtaattccgt 1020 actgctgccctcaagccctc ggggccacat tctctctggc gtggcaagca cggttttccc 1080 atcaccttaagttgcactta tttttcagct 1110 5 3157 DNA Murinae gen. sp. 5 aagctttgtgatattaatgg cagactgctc tccaggtcca actgtttgac tttttaattt 60 ttttaaatttgcgctctgct gagggactca ggccttggtg tatgcaagac aagtgtccct 120 gtcctggccacactcctagg cctgttgttt gttataaaag agatcacaag ggatacagac 180 caagcgaaaataaggggggg ctgtgagctt cattccctct cacatgatcc ctgcatatcc 240 agcaccctgcaaccagcttg ttcaaatctt gctcaagact gtaatagacc ttaatctgca 300 gtgaacacatccttccgggg agatgggtga gcagggttga aagtcacagt tcttcccatc 360 actaaggaaatcaaacagtt gcaaactcca tttcacatcc tatcagtggt ggagtacctt 420 aagattcacatacggttgat cgggagctgg gagaggaaca ggaactggcc tcacagctta 480 atgagacctctagaaagacg tttaaaggca gagggggtgt gtgaaaacaa gcaagggccc 540 tgatactccttggtaaggct aaacacaaat gcctgcggga tggcatggga gagggcagat 600 atggatgtaagctggcaagc catcagaagc tgagccgcac ccccctcccc ccatggtttg 660 ggagatggaagtggggcagg agaaaggccc atctgatagg ctgctatggc ggatagaccg 720 gcacggtgctggcatgtgct ttcacacaat atttactgaa ggcctgttgc ttgccaggag 780 ttgttcccggcgccggaaca gcaatgaaga aagtgaccag ggttttttct gagtctcaca 840 agttttccggtgaaggaggc ggaggatcga tgcatttcgg gctcctgctt ctgaggctga 900 ggagcatgtcgggttggcct ttctttgggg tgtcgcagca cacgtggagc cactcttgca 960 gggacatcgtgctgcgcgcg ccgccccggt ctcggtggcc tagccggctg gacgccgccc 1020 ccggcccagcctacacgtgg gttcccgcac gtccgctggg ctcccactct gacgtcagcg 1080 cggagcttccatttaaggcc ccgccgcgtg ctgctctgcg tgctggagcc actgtcgccg 1140 agctcggccacgctgcttct cctcgccagt cgccccccca tcgtgcatct agcggtacgg 1200 ttgggccccacgctgccggc acagggggtc ctatccgggg tggaggtgca gggtgttcag 1260 atttgggcacgcgtgggcta ctgtgctttg gggaacgtag caggcggccc acccagcctg 1320 ggcggcgacacccgtgtaaa gaggactaag ggtggctggc ctgaaggttg ggagccaccg 1380 aacgggggcgagggagcggt gcgaaacttg agctcccgta actcgagcca tgggggccga 1440 aaaagccttggcacgtccgg ggcgggtgct tcggagcagc cgtgcggttt gcattttcct 1500 ttgcgtgggctcggtggagt ggttgtttct gcagattata gggcgctctt gccctaaacg 1560 cattttgttaagaggctcgc tcctggtgtg actggggatc gtgctaaggc gctgcgtgcg 1620 gagaggaagcgggaagagtc gcagtccttc ctctgcaccc tagacggaag gaggaaacct 1680 gtagctgagaggcctgcgac acccatcggt cgtaggtggg aagtagaggc accctgtcct 1740 aatagagcacagacttgagg tttgcgtgaa gtagattctc tgagggaaga agtccccacg 1800 ccctgcccccagtcagcaga gacctcacaa ggctgtctag aaatagcagc ggttgtaagg 1860 agaccggattcctgcttgat ggccctagcg tgcttgccca gagatcttgt ccagtccttt 1920 tgcactttggaacgatttca aaaatagaca tggtgcttgc tgggaaaggt ggccatcgcg 1980 ggggtgggagtgggctccag gctcaggcct ccgcatattg tatccccatt ttggaactga 2040 agaaatttcccttagcggcc acatcctggg taatgaggcc ccgcctggtg gtgaaggccc 2100 taatcctgttagtggcattt tgagatctca ctctggcacc aggctaaggt ggctgccagc 2160 tccacttcaccgcgcttgtg ctttgggctc tagtaagggc caaactttga cgccgaactg 2220 cctgccagaggtctcatcca tggccagcag tcgctagacc tatatatatg cacctgattc 2280 tgaaattacgcgcactgcct tcccccgcct gccagtcttc tggatctggg ctggtcatac 2340 aacttgggttcttgcggggg tgggggggtt agaagaagct tgcgcgtgca ggcttaagca 2400 cgttgctatgccttggggtc gcaccttgtg gccgttattg gcgccctctg ctcttgattt 2460 ttggtacttcctggagcaac ttggcgctct acttgctgta gggctctggg tgatgggaga 2520 agagcgggagggcagctttc taaccatata agaggagata ccatcccctt ttggttcatc 2580 aagatgagtaagtcctcagg cggctacacg tacacggaga cctcggtatt atttttccat 2640 ttcaaggtagagccttcctg gtagagccag aaccacatcc tgccgctgct attcttggtt 2700 ttccacttctgttctttgtc acattgtcac ttaatggaaa ggagtccaag gtagcaagtc 2760 agcgtttttttttttttttt ttaaatacag ggacttggtg agtatacctt gggcaggtta 2820 caatgacacacggtgtatac cccttaggtc tcaaaagatt caaagtccaa gatggcaacc 2880 ctcaaggaccagctgattgt gaatcttctt aaggaagagc aggctcccca gaacaagatt 2940 acagttgttggggttggtgc tgttggcatg gcttgtgcca tcagtatctt aatgaaggta 3000 agtggagatcttcatggccc aagctatggg tgtggtgtgg gggagaggac atccctacat 3060 tgtcacattgtatgtaaaac tatcaaggtt tgcacacact cagtcatctg tgaaacattt 3120 tgcaacataatgatacacaa gaaagggatt atccaaa 3157

What is claimed is:
 1. A method for expressing a nucleic acid sequenceencoding a gene product in a tumour cell in vivo, comprising the stepsof: (a) providing a nucleic acid construct comprising ahypoxically-inducible expression control sequence comprising a nucleicacid sequence of CGTG, operatively linked to said nucleic acid sequenceencoding a gene product; (b) directly administering said nucleic acidconstruct to said tumour cell in vivo; and (c) exposing said tumour cellto hypoxic conditions, thereby expressing said nucleic acid sequenceencoding a gene product in said tumour cell in vivo under hypoxicconditions.
 2. The method according to claim 1, wherein thehypoxically-inducible expression control sequence is a phosphoglyceratekinase sequence comprising between about nucleotide −523 to aboutnucleotide −21 of the mouse PGK-1 gene which is nucleotide 423 tonucleotide 925 of SEQ ID No. 4, or a hypoxia-responsive fragmentthereof.
 3. The method according to claim 2, wherein thehypoxia-responsive fragment comprises the nucleic acid sequence of SEQID No.
 2. 4. The method according to claim 2, wherein thehypoxia-responsive fragment comprises the nucleic acid sequence of SEQID No.
 3. 5. The method according to claim 1, wherein saidhypoxically-inducible expression control sequence is isolated from theerythropoietin gene.
 6. The method according to claim 5, wherein thehypoxically-inducible expression control sequence comprises the nucleicacid sequence of SEQ ID No.
 1. 7. The method according to claim 1,wherein said hypoxically-inducible expression control sequence is alactate dehydrogenase sequence comprising between about nucleotide −186to about nucleotide +47 of the mouse LDH-A gene which is nucleotide 932to nucleotide 1164 of SEQ ID No. 5, or a hypoxia-responsive fragmentthereof.
 8. The method according to claim 1, wherein said nucleic acidconstruct comprises an additional one or more of thehypoxically-inducible expression control sequences.
 9. The methodaccording to claim 1, wherein said gene product is an enzyme whichconverts a prodrug to an active drug.
 10. The method according to claim9, wherein said enzyme is cytosine deaminase.
 11. The method accordingto claim 1, wherein said gene product is a cytokine.
 12. A method forexpressing a nucleic acid sequence encoding a gene product in amammalian cell in vivo, comprising the steps of: (a) providing a nucleicacid construct comprising a hypoxically-inducible expression controlsequence comprising a nucleic acid sequence of CGTG, operatively linkedto said nucleic acid sequence encoding a gene product; (b) directlyadministering said nucleic acid construct to said mammalian cell invivo; and (c) exposing said mammalian cell to hypoxic conditions,thereby expressing said nucleic acid sequence encoding a gene product insaid mammalian cell in vivo under hypoxic conditions.
 13. The methodaccording to claim 12, wherein the hypoxically-inducible expressioncontrol sequence is a phosphoglycerate kinase sequence comprisingbetween about nucleotide −523 to about nucleotide −21 of the mouse PGK-1gene which is nucleotide 423 to nucleotide 925 of SEQ ID No. 4, or ahypoxia-responsive fragment thereof.
 14. The method according to claim13, wherein the hypoxia-responsive fragment comprises the nucleic acidsequence of SEQ ID No.
 2. 15. The method according to claim 13, whereinthe hypoxia-responsive fragment comprises the nucleic acid sequence ofSEQ ID No.
 3. 16. The method according to claim 12, wherein thehypoxically-inducible expression control sequence is isolated from theerythropoietin gene.
 17. The method according to claim 16, wherein saidhypoxically-inducible expression control sequence comprises SEQ IDNo.
 1. 18. The method according to claim 12, wherein saidhypoxically-inducible control sequence is a lactate dehydrogenasesequence comprising between about nucleotide −186 to about nucleotide+47 of the mouse LDH-A gene which is nucleotide 932 to nucleotide 1164of SEQ ID No. 5, or a hypoxia-responsive fragment thereof.
 19. Themethod according to claim 12, wherein said mammalian cell is a tumourcell.
 20. The method according to claim 12, wherein said gene product isan enzyme which converts a prodrug to an active drug.
 21. The methodaccording to claim 20, wherein said enzyme is cytosine deaminase. 22.The method according to claim 12, wherein said gene product is acytokine.
 23. A nucleic acid construct comprising ahypoxically-inducible expression control sequence comprising a nucleicacid sequence of CGTG operatively linked to a heterologous nucleic acidsequence encoding a gene product, wherein said gene product is expressedunder hypoxic conditions when said nucleic acid construct is present ina mammalian cell, and wherein said nucleic acid construct does notcomprise an erythropoietin hypoxically-inducible expression controlsequence.
 24. The nucleic acid construct according to claim 23, whereinthe hypoxically-inducible expression control sequence is aphosphoglycerate kinase sequence comprising between about nucleotide−523 to about nucleotide −21 of the mouse PGK-1 gene which is nucleotide423 to nucleotide 925 of SEQ ID No. 4, or a hypoxia-responsive fragmentthereof.
 25. The nucleic acid construct according to claim 24, whereinthe hypoxia-responsive fragment of the phosphoglycerate kinase sequencescomprises the nucleic acid sequence of SEQ ID No.
 2. 26. The nucleicacid construct according to claim 24, wherein the hypoxia-responsivefragment of the phosphoglycerate kinase sequence comprises the nucleicacid sequence of SEQ ID No.
 3. 27. The nucleic acid construct accordingto claim 23, wherein said mammalian cell is a tumour cell.
 28. Thenucleic acid construct according to claim 23, wherein saidhypoxically-inducible expression control sequence is a lactatedehydrogenase sequence comprising between about nucleotide −186 to aboutnucleotide +47 of the mouse LDH-A gene which is nucleotide 932 tonucleotide 1164 of SEQ ID No. 5, or a hypoxia-responsive fragmentthereof.
 29. The nucleic acid construct of claim 23, wherein saidnucleic acid construct comprises two or more of thehypoxically-inducible expression control sequences.
 30. The nucleic acidconstruct according to claim 23, wherein said gene product is an enzymewhich converts a prodrug to an active drug.
 31. The nucleic acidconstruct according to claim 30, wherein said enzyme is cytosinedeaminase.
 32. The nucleic acid construct according to claim 23, whereinsaid gene product is a cytokine.
 33. A nucleic acid construct comprisingthree or more hypoxically-inducible expression control sequences, eachof said hypoxically-inducible expression control sequences comprising anucleic acid sequence of CGTG, wherein said hypoxically-inducibleexpression control sequences are operatively linked to a heterologousnucleic acid sequence encoding a gene product, and wherein said geneproduct is expressed when present in a mammalian cell under hypoxicconditions.
 34. The nucleic acid construct according to claim 33,wherein at least one hypoxically-inducible expression control sequenceis a phosphoglycerate kinase sequence comprising between aboutnucleotide −523 to about nucleotide −21 of the mouse PGK-1 gene which isnucleotide 423 to nucleotide 925 of SEQ ID No. 4, or ahypoxia-responsive fragment thereof.
 35. The nucleotide acid constructaccording to claim 34, wherein the hypoxia-responsive fragment of thephosphoglycerate kinase sequence comprises the nucleic acid sequence ofSEQ ID No.
 2. 36. The nucleic acid construct according to claim 34,wherein the hypoxia-responsive fragment of the phosphoglycerate kinasesequence comprises the nucleic acid sequence of SEQ ID No.
 3. 37. Thenucleic acid construct according to claim 33, wherein at least onehypoxically-inducible expression control sequence is isolated from theerythropoietin gene.
 38. The nucleic acid construct according to claim37, wherein the at least one hypoxically-inducible expression controlsequence comprises the nucleic acid sequence of SEQ ID No.
 1. 39. Thenucleic acid construct according to claim 33, wherein at least onehypoxically-inducible expression control sequence is a lactatedehydrogenase sequence comprising between about nucleotide −186 to aboutnucleotide +47 of the mouse LDH-A gene which is nucleotide 932 tonucleotide 1164 of SEQ ID No. 5, or a hypoxia-responsive fragmentthereof.
 40. The nucleic acid construct according to claim 33, whereinsaid gene product is an enzyme which converts a prodrug to an activedrug.
 41. The nucleic acid construct according to claim 40, wherein saidenzyme is cytosine deaminase.
 42. The nucleic acid construct accordingto claim 33, wherein said gene product is a cytokine.
 43. A tumour cellcomprising a nucleic acid construct comprising a hypoxically-inducibleexpression control sequence comprising a nucleic acid sequence of CGTGoperatively linked to a heterologous nucleic acid sequence encoding agene product, wherein said gene product is expressed in said tumour cellunder hypoxic conditions, and wherein said nucleic acid construct doesnot comprise an erythropoietin hypoxically-inducible expression controlsequence.
 44. The tumour cell according to claim 43, wherein thehypoxically-inducible expression control sequence is a phosphoglyceratekinase sequence comprising between about nucleotide −523 to aboutnucleotide −21 of the mouse PGK-1 gene which is nucleotide 423 tonucleotide 925 of SEQ ID No. 4, or a hypoxia-responsive fragmentthereof.
 45. The tumour cell according to claim 44, wherein thehypoxia-responsive fragment of the phosphoglycerate kinase sequencescomprises the nucleic acid sequence of SEQ ID No.
 2. 46. The tumour cellaccording to claim 44, wherein the hypoxia-responsive fragment of thephosphoglycerate kinase sequence comprises the nucleic acid sequence ofSEQ ID No.
 3. 47. The tumour cell according to claim 43, wherein saidhypoxically-inducible expression control sequence is a lactatedehydrogenase sequence comprising between about nucleotide −186 to aboutnucleotide +47 of the mouse LDH-A gene which is nucleotide 932 tonucleotide 1164 of SEQ ID No. 5, or a hypoxia-responsive fragmentthereof.
 48. The tumour cell according to claim 43, wherein said nucleicacid construct comprises two or more of the hypoxically-inducibleexpression control sequences.
 49. The tumour cell according to claim 43,wherein said gene product is an enzyme which converts a prodrug to anactive drug.
 50. The tumour cell according to claim 49, wherein saidenzyme is cytosine deaminase.
 51. The tumour cell according to claim 43,wherein said gene product is a cytokine.
 52. A mammalian cell comprisinga nucleic acid construct comprising a hypoxically-inducible expressioncontrol sequence comprising a nucleic acid sequence of CGTG operativelylinked to a heterologous nucleic acid sequence encoding a gene product,wherein said gene product is expressed in said mammalian cell underhypoxic conditions, and wherein said nucleic acid construct does notcomprise an erythropoietin hypoxically-inducible expression controlsequence.
 53. The mammalian cell according to claim 52, wherein thehypoxically-inducible expression control sequence is a phosphoglyceratekinase sequence comprising between about nucleotide −523 to aboutnucleotide −21 of the mouse PGK-1 gene which is nucleotide 423 tonucleotide 925 of SEQ ID No. 4, or a hypoxia-responsive fragmentthereof.
 54. The mammalian cell according to claim 53, wherein thehypoxia-responsive fragment of the phosphoglycerate kinase sequencescomprises the nucleic acid sequence of SEQ ID No.
 2. 55. The mammaliancell according to claim 53, wherein the hypoxia-responsive fragment ofthe phosphoglycerate kinase sequence comprises the nucleic acid sequenceof SEQ ID No.
 3. 56. The mammalian cell according to claim 52, whereinsaid hypoxically-inducible expression control sequence is a lactatedehydrogenase sequence comprising between about nucleotide −186 to aboutnucleotide +47 of the mouse LDH-A gene which is nucleotide 932 tonucleotide 1164 of SEQ ID No. 5, or a hypoxia-responsive fragmentthereof.
 57. The mammalian cell according to claim 52, wherein saidnucleic acid construct comprises two or more hypoxically-inducibleexpression control sequences.
 58. The mammalian cell according to claim52, wherein said gene product is an enzyme which converts a prodrug toan active drug.
 59. The mammalian cell according to claim 58, whereinsaid enzyme is cytosine deaminase.
 60. The mammalian cell according toclaim 52, wherein said gene product is a cytokine.
 61. A method forenhancing the expression of a nucleic acid sequence encoding a geneproduct in a tumour cell in vivo, comprising the steps of: (a) providinga nucleic acid construct comprising two or more hypoxically-inducibleexpression control sequences, each of said hypoxically-inducibleexpression control sequences comprising a nucleic acid sequence of CGTGwherein said hypoxically-inducible expression control sequences areoperatively linked to said nucleic acid sequence encoding a geneproduct; (b) directly administering said nucleic acid construct to saidtumour cell in vivo; and (c) exposing said tumour cell to hypoxicconditions, thereby enhancing expression of said nucleic acid encoding agene product in said tumour cell in vivo under hypoxic conditions incomparison with expression of the same nucleic acid encoding a geneproduct in operative linkage with only one of the hypoxically-inducibleexpression control sequences.
 62. The method according to claim 61,wherein at least one hypoxically-inducible expression control sequenceis a phosphoglycerate kinase sequence comprising between aboutnucleotide −523 to about nucleotide −21 of the mouse PGK-1 gene which isnucleotide 423 to nucleotide 925 of SEQ ID No. 4, or ahypoxia-responsive fragment thereof.
 63. The method according to claim62, wherein the hypoxia-responsive fragment comprises the nucleic acidsequence of SEQ ID No.
 2. 64. The method according to claim 62, whereinthe hypoxia-responsive fragment comprises the nucleic acid sequence ofSEQ ID No.
 3. 65. The method according to claim 61, wherein at least onehypoxically-inducible expression control sequence is isolated from theerythropoietin gene.
 66. The method according to claim 65, wherein theat least one hypoxically-inducible expression control sequence comprisesthe nucleic acid sequence of SEQ ID No.
 1. 67. The method according toclaim 61, wherein at least one hypoxically-inducible expression controlsequence is a lactate dehydrogenase sequence comprising between aboutnucleotide −186 to about nucleotide +47 of the mouse LDH-A gene which isnucleotide 932 to nucleotide 1164 of SEQ ID No. 5, or ahypoxia-responsive fragment thereof.
 68. The method according to claim61, wherein said nucleic acid construct comprises an additional one ormore hypoxically-inducible expression control sequences.
 69. The methodaccording to claim 61, wherein said gene product is an enzyme whichconverts a prodrug to an active drug.
 70. The method according to claim69, wherein said enzyme is cytosine deaminase.
 71. The method accordingto claim 61, wherein said gene product is a cytokine.
 72. A method forenhancing the expression of a nucleic acid sequence encoding a geneproduct in a mammalian cell in vivo, comprising the steps of: (a)providing a nucleic acid construct comprising two or morehypoxically-inducible expression control sequences, each of saidhypoxically-inducible expression control sequences comprising a nucleicacid sequence of CGTG, wherein said hypoxically-inducible expressioncontrol sequences are operatively linked to said nucleic acid sequenceencoding a gene product; (b) directly administering said nucleic acidconstruct to said mammalian cell in vivo; and (c) exposing saidmammalian cell to hypoxic conditions, thereby enhancing expression ofsaid nucleic acid encoding a gene product in said mammalian cell in vivounder hypoxic conditions in comparison with expression of the samenucleic acid encoding a gene product in operative linkage with only oneof the hypoxically-inducible expression control sequences.
 73. Themethod according to claim 72, wherein at least one hypoxically-inducibleexpression control sequence is a phosphoglycerate kinase sequencecomprising between about nucleotide −523 to about nucleotide −21 of themouse PGK-1 gene which is nucleotide 423 to nucleotide 925 of SEQ ID No.4, or a hypoxia-responsive fragment thereof.
 74. The method according toclaim 73, wherein the hypoxia-responsive fragment comprises the nucleicacid sequence of SEQ ID No.
 2. 75. The method according to claim 73,wherein the hypoxia-responsive fragment comprises the nucleic acidsequence of SEQ ID No.
 3. 76. The method according to claim 72, whereinat least one hypoxically-inducible expression control sequence isisolated from the erythropoietin gene.
 77. The method according to claim76, wherein the at least one hypoxically-inducible expression controlsequence comprises the nucleic acid sequence of SEQ ID No.
 1. 78. Themethod according to claim 72, wherein at least one hypoxically-inducibleexpression control sequence is a lactate dehydrogenase sequencecomprising between about nucleotide −186 to about nucleotide +47 of themouse LDH-A gene which is nucleotide 932 to nucleotide 1164 of SEQ IDNo. 5, or a hypoxia-responsive fragment thereof.
 79. The methodaccording to claim 72, wherein said nucleic acid construct comprises anadditional one or more hypoxically-inducible expression controlsequences.
 80. The method according to claim 72, wherein said geneproduct is an enzyme which converts a prodrug to an active drug.
 81. Themethod according to claim 80, wherein said enzyme is cytosine deaminase.82. The method according to claim 72, wherein said gene product is acytokine.